Analysis Illumina data
Heleen
8/6/2020
1. Pre blast
Load table and check read numbers
Functions:
getData <- function(){
otu <- fread("~/Documents/derep_illum/changedheader/otu.about")
before <- nrow(otu)
# Remove X. or X from colnames
names(otu) <- sub("#", "", names(otu))
otu <- column_to_rownames(otu, var = "OTU ID")}
# optional
remove_chimeras <- function(){
chimeras <- read.csv("~/Documents/IlluminaAdaptertrimmedAllreps/thingremoved", header=FALSE, sep=";")
otu <- column_to_rownames(otu, var = "OTU.ID")
otu <- otu[ ! sub("^.*?:", "", otu$OTU.ID) %in% chimeras$V1,] ##remove all chimeras
after <- nrow(otu)
cat(paste("removed", before-after, "chimeric sequences\n\n"))
rownames(otu) <- NULL
return(otu)}
# optional chimera removal: otu <- remove_chimeras()
# print results nicely:
myOTUcat <- function(){
#total read sum in all clusters
total_reads <- sum(rowSums(otu))
cat(paste('total reads (grand total with which clustering was done):\n',
total_reads))
cat("\n\n", 'Summary statistics of number of reads per OTU:\n')
print(summary(rowSums(otu)))
cat(paste("\n\n",
'Total number of OTUs (including singletons):\n', nrow(otu)))
}Execution:
otu <- getData()
myOTUcat()## total reads (grand total with which clustering was done):
## 2512237
##
## Summary statistics of number of reads per OTU:
## Min. 1st Qu. Median Mean 3rd Qu. Max.
## 1.00 1.00 1.00 8.46 2.00 86074.00
##
##
## Total number of OTUs (including singletons):
## 296884Prepare Saba location data
Functions:
#function to change decimal to comma in one
decimal_to_comma <- function(data, column){
data[,column] <- sub(",", ".",
data[,column],
fixed = TRUE)}
prepLocSaba <- function(){
## load the Saba sample location data
locdata_saba <- read.delim("~/Downloads/NICO5-eDNA-64PE432-Metadata-MinIon - DataFilterSaba.txt")
## Change samplenames, colnames in metadatafile so they match the OTU file making merging is possible.
## Change decimal to comma for computation.
locdata_saba[,1] <- gsub("(?<![0-9])0+", "", locdata_saba[,1], perl = TRUE)
locdata_saba[,1] <- gsub("\\.", "_", locdata_saba[,1], perl = TRUE)
locdata_saba[,1] <- tolower(locdata_saba[,1])
## change long colnams to lat,long, altitude
names(locdata_saba)[names(locdata_saba)=="geo_lat..in.decimalen..WGS84."] <- "lat"
names(locdata_saba)[names(locdata_saba)=="geo_lon..in.decimalen..WGS84."] <- "long"
names(locdata_saba)[names(locdata_saba)=="altitude..in.meters.aasl."] <- "altitude"
names(locdata_saba)[1] <- "sample"
## change decimals to commas
for (col in c("lat", "long")){
locdata_saba[, col] <- as.numeric(decimal_to_comma(locdata_saba, col))}
return(locdata_saba)}Execution:
# establish samples and controls
controls <- c("sxm_2018_62", "sxm_2018_63", "sxm_2018_64",
"sxm_2018_65", "sxm_2018_66", "sxm_2018_70",
"sxm_2018_71", "0", "unicon1",
"unicon1A", "neg_controle")
samples <- names(otu)[-which(names(otu) %in% controls)]
locdata_saba <- prepLocSaba()
# take only the columns needed, and the samplenames needed
locdata_saba <- locdata_saba %>%
select(sample, lat, long, altitude, habitat) %>%
filter(sample %in% samples)load and prepare Statia data
select only relevant columns and rows, and setnames, and change the numbers to depth
# take relevant columns, take out the samples that are not in the OTU table, and set the colnames to same names as Saba data.
locdata_statia <- read.delim("~/statia_location.txt") %>%
select(Field.nr., lat, long, Average.depth) %>% # select relevant columns
filter(!Field.nr. %in% c(528, 529)) %>% # discard irrelevant rows
setNames(c("sample", "lat", "long", "altitude")) %>% # change column names
mutate(altitude = as.numeric(gsub('[+]', '', altitude)) * -1) # mutate altidue column to negativeBind Saba and statia data by row (to get merged data frame (mdf))
mdf <- plyr::rbind.fill(locdata_saba, locdata_statia)estimate boxcore altitude data
boxcore altitude data is missing, so it’s estimated by taking the nearest point geographically of which altitude data is available
#fill in missing boxcore altitude data wth nearestby latitude , the lowest value of that
mdf <- mdf %>%
group_by(lat) %>%
# arrange the groups by descending altitude within the groups
arrange(desc(altitude), .by_group = TRUE) %>%
# make new column with lowest altitude of group if the value is missing
mutate(altitude = ifelse(is.na(altitude), min(altitude, na.rm = TRUE), altitude)) %>%
# because for some boxcore samples it was taken at a slgihty different latitude, it does not belong to a group.
# Thus, R introduces infinite values which this command changes to NA values
mutate(altitude = ifelse(is.infinite(altitude), NA, altitude)) %>%
# needs to be ungrouped to fill it with the nearest & lowest altitude
ungroup() %>%
fill(altitude, .direction = 'down')## Warning in min(altitude, na.rm = TRUE): no non-missing arguments to min;
## returning Inf
## Warning in min(altitude, na.rm = TRUE): no non-missing arguments to min;
## returning InfPut every sample in north, south or statia catogery based on latitude, to enable exchange testing. Add a tag indication what region the sampling location is in: Saba north, Saba south, or Statia.
mdf$tag <- ifelse(grepl("^[0-9]+$", mdf$sample), 'Statia',
ifelse(mdf$lat > 17.55, "Saba North",
"Saba South")) %>% as.factor()Control reads
investigate control reads
####Functions:
#prep data
controlDf <- function(){
copy <- otu
copy[copy>0] <- 1
per_type <- copy %>% colSums() %>%
as.data.frame() %>% rownames_to_column(var = "sample")%>%
mutate(type = ifelse(sample %in% bottlecontrol, "storage bottle control",
ifelse(grepl("unicon", sample), "pcr + control",
ifelse(sample %in% c("0", "neg_controle"),
"pcr - control", "samples")))) %>%
mutate(type = fct_reorder(type, desc(.)))
return(per_type)}
plot_pertype <- function(df){
control_plotOTU <-
ggplot(df, aes(x = type,
y = .,
fill = type)) +
geom_boxplot() +
labs(title = "Number of OTUs per sample type",
subtitle = "before abundance filterig",
x = "Type of sample",
y = "OTUs (log10)") +
scale_fill_jco(alpha = 0.6) +
scale_y_log10() +
# edit lines and background
theme(text = element_text(size = 20),
panel.grid.major.x = element_blank(),
panel.grid.major.y = element_line("gray50", size = 0.2),
panel.background = element_blank(),
axis.line = element_line("gray50"),
legend.position = "none")
control_plotOTU}Execution:
controls <- c("sxm_2018_62", "sxm_2018_63", "sxm_2018_64",
"sxm_2018_65", "sxm_2018_66", "sxm_2018_70",
"sxm_2018_71", "0", "unicon1",
"unicon1A", "neg_controle")
bottlecontrol <- c("sxm_2018_62", "sxm_2018_63", "sxm_2018_64",
"sxm_2018_65", "sxm_2018_66", "sxm_2018_70",
"sxm_2018_71")
controlDf <- controlDf()
plot_pertype(controlDf)
anova of storage bottle control
res.aov <- aov(d = controlDf, . ~ type)
summary(res.aov)## Df Sum Sq Mean Sq F value Pr(>F)
## type 3 1.763e+08 58764044 3.444 0.0198 *
## Residuals 96 1.638e+09 17060989
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1TukeyHSD(res.aov)## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = . ~ type, data = controlDf)
##
## $type
## diff lwr upr p adj
## storage bottle control-samples -3137.205 -7376.563 1102.152 0.2204976
## pcr + control-samples -5496.348 -13218.159 2225.463 0.2517563
## pcr - control-samples -5778.848 -13500.659 1942.963 0.2117674
## pcr + control-storage bottle control -2359.143 -11018.102 6299.817 0.8919758
## pcr - control-storage bottle control -2641.643 -11300.602 6017.317 0.8553299
## pcr - control-pcr + control -282.500 -11082.120 10517.120 0.9998844# check for assumptions
check_assumption <- function(){
plot(res.aov, 1) # homogeneity of variances
plot(res.aov, 2) # normality of residuals
shapiro.test(residuals(res.aov))} # shapiro wilk of anova residualsPost-blast
investigate storage bottle control identifications
`lca storage` <- read.delim("~/Documents/derep_illum/controls/underep/taxadded/lca") ## load lca file of storage bottle control blast
species <- table(`lca storage`$X.genus) %>%
data.frame() %>%
mutate(Var1 = ifelse(Freq < 1000, "Other", as.character(Var1))) %>%
filter(!Var1=="no identification") %>%
group_by(Var1) %>%
dplyr::summarise(Freq = sum(Freq)) %>%
mutate(Prop = (Freq/sum(Freq))*100) %>%
ungroup() %>%
mutate(Var1 = fct_reorder(Var1, desc(Freq))) %>%
mutate(Var1 = fct_relevel(Var1, "Other", after = Inf))## `summarise()` ungrouping output (override with `.groups` argument)get_col <- function(){
colorcount <- length(genuscount$Var1)
qual_col <- brewer.pal.info[brewer.pal.info$category == "qual",]
col_vector <- unlist(mapply(brewer.pal,
qual_col$maxcolors, rownames(qual_col)))
mycol <- sample(col_vector, colorcount)}
ggplot(species, aes(x = Var1, y = Freq, fill = Var1)) +
geom_bar(stat = "identity", color = "black") +
theme(text = element_text(size = 20),
axis.text.x = element_text(angle = 45, hjust =1, size = 15),
legend.position = "none") +
scale_fill_jco() +
labs(title = "Genera represented in storage bottles",
x = "Genus\n" ,
y = "Number of identifications (log10)") +
scale_y_log10()
# Pie Chart
# add position of label
count.data <- species %>%
arrange(desc(Var1)) %>%
mutate(lab.ypos = cumsum(Prop) - 0.5*Prop)
ggplot(count.data, aes(x = "", y = Prop, fill = Var1)) +
geom_bar(width = 1, stat = "identity", color = "white") +
coord_polar("y", start = 0)+
geom_text(aes(y = lab.ypos, label = round(Prop,1)), color = "white")+
theme_void()
Filter out out controls
If a OTU also contains control reads, these need to be filtered out of the samples contain them in frequencies that are close to the control frequencies. This could be contamination from the bottles the sample was stored in, or PCR contamination.
posContamination <- function(){
contam <- otu %>% filter(unicon1 > 5000) %>% select(!c("unicon1",
"unicon1A")) %>% sum()
total <- otu %>% filter(unicon1 > 5000) %>% sum()
freq <- contam/total
cat(paste("Contamination percentage of positive control in other samples: \t", round(freq*100, 5), "\n"))
return(freq)}
negContamination <- function(){
contam <- otu %>% select(neg_controle) %>% sum()
total <- otu %>% filter(neg_controle>0) %>% sum()
freq <- contam/total
cat(paste("Contamination percentage in negative samples: \t", round(freq*100, 5)))}
rate <- posContamination()## Contamination percentage of positive control in other samples: 0.00671negContamination()## Contamination percentage in negative samples: 0.0612Low abundance filter
the rate of contamination in the positive control was used as low abundance filter rate.
lowAbuncanceFilter <- function(rate){
before <- nrow(otu)
colsum <- colSums(otu)
min_read <- colsum * rate # if OTU contains less than this many reads, filter out
otu <-
mapply(col = otu, min = min_read, function(col, min){
col[col < min] <- 0
col}) %>%
as.data.frame () %>%
`rownames<-`(rownames(otu)) %>% filter(!rowSums(.[samples]) == 0) # take out "empty" otus
after <- nrow(otu)
percenage_ret <- ((before-after)/before)*100
cat(paste("filtered out ", before-after, " OTUs, which is ",
round(percenage_ret, 2), "% of original OTUs
\n\n",
after, " OTUs were retained", sep = ""))
return(otu)
}
controls <- c("sxm_2018_62", "sxm_2018_63", "sxm_2018_64",
"sxm_2018_65", "sxm_2018_66", "sxm_2018_70",
"sxm_2018_71", "0", "unicon1",
"unicon1A", "neg_controle")
bottlecontrol <- c("sxm_2018_62", "sxm_2018_63", "sxm_2018_64",
"sxm_2018_65", "sxm_2018_66", "sxm_2018_70",
"sxm_2018_71")
samples <- names(otu)[-which(names(otu) %in% controls)]
otu <- lowAbuncanceFilter(rate = rate)## filtered out 244221 OTUs, which is 82.26% of original OTUs
##
##
## 52663 OTUs were retainedremove singleton OTUs
remove_singletons <- function(){
OTUbefore <- nrow(otu)
otu <- otu %>% filter(!rowSums(.[samples]) < 2) # remove all rows where rowSum == 1 (singleton OTU)
OTUafter <- nrow(otu)
removed <- OTUbefore-OTUafter
retained_percent <- round(OTUafter/OTUbefore*100, 2)
cat(paste("removed", removed, "singletons\n",
"retained", OTUafter, "OTUs, which means", retained_percent, "percent of OTUs was retained",
sep = " "))
return(otu)}
otu <- remove_singletons()## removed 12254 singletons
## retained 40409 OTUs, which means 76.73 percent of OTUs was retainedplot number of otus per sample
saba <- samples[grepl("sxm", samples)]
copy <- otu
copy[copy>0] <- 1
copy <- copy %>% colSums() %>%
as.data.frame() %>% rownames_to_column(var = "sample")%>%
mutate(type = ifelse(sample %in% bottlecontrol, "storage bottle control",
ifelse(grepl("unicon", sample), "pcr + control",
ifelse(sample %in% c("0", "neg_controle"),
"pcr - control", "samples")))) %>%
mutate(type = fct_reorder(type, desc(.)))
control_plotOTU <-
ggplot(copy, aes(x = type,
y = .,
fill = type)) +
geom_boxplot() +
labs(title = "Number of OTUs per sample type",
subtitle = "After abundance filter",
x = "Type of sample",
y = "OTUs (log10)") +
scale_fill_jco(alpha = 0.6) +
scale_y_log10() +
# edit lines and background
theme(text = element_text(size = 20),
panel.grid.major.x = element_blank(),
panel.grid.major.y = element_line("gray50", size = 0.2),
panel.background = element_blank(),
axis.line = element_line("gray50"),
legend.position = "none")
control_plotOTU## Warning: Transformation introduced infinite values in continuous y-axis## Warning: Removed 1 rows containing non-finite values (stat_boxplot).
ggsave("controlplot AFTER abundance filter", plot = control_plotOTU, device = "png", height = 7, width = 10)## Warning: Transformation introduced infinite values in continuous y-axis
## Warning: Removed 1 rows containing non-finite values (stat_boxplot).Additional bottle control contamination check for saba samples
And remove controls from otu table
# establish controls and samples
controls <- c("sxm_2018_62", "sxm_2018_63", "sxm_2018_64",
"sxm_2018_65", "sxm_2018_66", "sxm_2018_70",
"sxm_2018_71", "0", "unicon1",
"unicon1A", "neg_controle")
bottlecontrol <- c("sxm_2018_62", "sxm_2018_63", "sxm_2018_64",
"sxm_2018_65", "sxm_2018_66", "sxm_2018_70",
"sxm_2018_71")
samples <- names(otu)[-which(names(otu) %in% controls)]
saba <- samples[grepl("sxm", samples)]
# go over the rows(OTUs) where there are control reads and change any reads to 0 if they contain less than twic the number of control reads
filter_controls <- function(){
OTUbefore <- nrow(otu)
mcr <- do.call(pmax, otu[bottlecontrol]) # max control value for each otu
mcp <- mcr > 0 # control values > 0
otu[mcp, saba][otu[mcp, saba] < 2*mcr[mcp]] <- 0
# discard controls, and OTUs that have no reads associated bc of control filter
otu <- otu %>%
select(all_of(samples)) %>% #only keep samples
filter(!rowSums(.) == 0)# discard OTUs that have no reads because of filtering
ncolbefore <- ncol(otu)
OTUafter <- nrow(otu)
cat(paste("Control filtering removed", OTUbefore-OTUafter, "OTUs, which is ",
round(((OTUbefore-OTUafter)/OTUbefore)*100, 2)), "%")
otu <- otu[,colSums(otu) > 2000]
ncolafter <- ncol(otu)
after2000 <- nrow(otu)
cat(paste("\n\nanother", OTUafter-after2000, "OTUs, were removed by removing ", ncolbefore-ncolafter, "samples that head read numbers below 2000 reads"))
return(otu)}
otu <- filter_controls()## Control filtering removed 33 OTUs, which is 0.08 %
##
## another 0 OTUs, were removed by removing 3 samples that head read numbers below 2000 reads# select only samples that have read counts of higher than two thousandwrite sequences to blast to file
now that all control reads and singletons have been filtered out, the remaining OTUs can be blasted. For this, the OTU centroid sequences from filtering step at 98% are extracted and then blasted -> taxadded -> dummyadded(for LCA script to work) -> lca script. Then its back to R
# make file with which sequences to be blasted can be selected (singletons filtered out)
write.table(rownames(otu),
file = '~/OTUcentroids.txt',
row.names = FALSE,
quote = FALSE,
col.names = FALSE)
nrow(otu)## [1] 403763. LCA
remove bacteria hits
The entire dataset was blasted to genbank in galaxy. The resulting lca file is imported here to remova any reads belonging to bacteria.
genbank <- read.delim("~/Downloads/Galaxy6-[filtOTUseqs40376.fasta_BLAST_original_taxonomy_lca].tabular")
bact <- genbank %>% filter(X.kingdom == "Bacteria")
toremove <- bact$X.Query
length(toremove)## [1] 22428otu <- otu[!row.names(otu) %in% toremove,]
nrow(otu)## [1] 17948import and prepare lca data
Then, the lca files at bitscore 8 and 12 percent are imported and compared
getLca <- function(){
df <- read.delim("~/Documents/derep_illum/changedheader/taxadded/bit8range")
df2 <- read.delim("~/Documents/derep_illum/changedheader/taxadded/bit12range")
dfs <- list(df, df2)
# remove X. from cols and name the dfs
dfs <-
lapply(dfs, function(x){setNames(x, sub("^X.", "", names(x)))}) %>%
`names<-`(c("Bitscore = 8", "Bitscore = 12"))}
#execute the functions
lcas <- getLca()
lcas <- lapply(lcas, function(x) {x <- x[!x$Query %in% toremove,]})
# add information on how many reads were captured by the bitscore threshold
merged_dfs <-
lapply(1:length(lcas), function(x) lcas[[x]] %>%
data.frame) %>%
# create extra column with what bitscore was used and bind the dataframes
Map(cbind, ., Bitscore_setting = names(lcas)) %>% # info of bitscore for bth dfs
do.call(rbind, .) %>% #combined them by row
data.frame() %>%
filter(!grepl("sp\\.", species)) # remove hits that contain sp. because theyre not informative.
# get factor levels in right order for nice looking plots
merged_dfs$lca.rank <- factor(merged_dfs$lca.rank, levels = c("no identification", colnames(merged_dfs)[4:10])) plot number of taxa found per rank by bitscore setting
Bitscore 8 has a stronger bias towards genus level identifications because the it takes fewer reads into account for the lca deterimination so higher chance that theres only 1 read to do taxa determination with,
myPlot <- function(){
merged_dfs %>%
ggplot(aes(x= lca.rank, y = ..count.., group = Bitscore_setting)) +
geom_bar(aes(fill=`Bitscore_setting`),
position = "dodge", alpha = 0.8) +
geom_text(stat = "count",
aes(label = ..count.., colour = Bitscore_setting),
position = position_dodge(0.9),
vjust = -0.5, size = 3) +
scale_fill_jco() +
#scale_fill_brewer(type = "qual", palette = "Pastel2") +
#scale_fill_manual(values = alpha(c("#00AFBB", "#FC4E07"), 0.8)) +
theme_minimal() +
scale_color_manual(values = c("gray30", "gray8"),
guide = F) +
scale_y_log10() +
labs(title = "Number of LCA identifications per rank",
subtitle = "by bitscore setting",
x = "Rank",
y = "Count (log10)",
fill = "Bitscore setting") +
theme(plot.title = element_text(size = 15, vjust = 1),
text = element_text(size = 20),
axis.text.x = element_text(angle = 45, hjust =1, size = 15))}
p <- myPlot()
p
ggsave(filename = "Bitscore plot", p, device = "png", width = 10, height = 7.5)The plot shows that bitscore 12 has less identifications but also less of a bias toward genus level, at it takes more reads into the LCA step.
Combine OTU and sampling location data
Compare the number of centroids supplied to the blast file to the number of blast hits found that had at least one blast hit of 70% identity and 70% coverage. The LCA file wiht bitscore 12 is chosen. get LCA: numbers
merge_otu_lca <- function(){
otu <- rownames_to_column(otu, var = "Query")
df_otu_lca <- merge(otu, lcas[[2]], by='Query', all.x = TRUE)
total_otu <- nrow(otu)
OTUs_hit <- length(unique(lcas[[2]]$Query))
lca_hit <- length(lcas[[2]]$lca.rank[lcas[[2]]$lca.rank != "no identification"])
cat(paste("\tnumber of rows in OTU file (number of OTUs found):\t", total_otu,
"\n\n",
"\tnumber of rows in LCA file (OTUs that had at least one blast hit):\t", OTUs_hit,
"\n\nPercentage of dark taxa is: ", round((1-lca_hit/total_otu)*100,2), "%"))
return(df_otu_lca)}
otu_lca <- merge_otu_lca() ## number of rows in OTU file (number of OTUs found): 17948
##
## number of rows in LCA file (OTUs that had at least one blast hit): 3848
##
## Percentage of dark taxa is: 81.87 %Combine sampling location data with the OTU table
Functions
get_bin_tags <- function(df){
mdf %>%
filter(sample %in% colnames(df)) %>%
mutate(habitat = as.character(habitat)) %>%
mutate(habitat = replace_na(habitat, "Mixed")) %>%
mutate(bin = paste(tag, habitat)) %>%
t() %>%
as.data.frame() %>%
row_to_names(1) %>%
rownames_to_column(var = "Query") %>%
filter(Query == "bin")
}
get_tags <- function(df){
mdf %>%
filter(sample %in% colnames(df)) %>%
t() %>%
as.data.frame() %>%
row_to_names(1) %>%
rownames_to_column(var = "Query") %>%
filter(Query == "tag")}
replace_colnames <- function(df){
df <- df %>% rownames_to_column(var = "Query")
with_bins <- rbind.fill(tags, df) %>%
row_to_names(row_number = 1)
colnames(with_bins)[1] <- "Query"
return(with_bins)
}
# make a new df with one column calles Query (for merging), bind by col and remove rownames
#otu <- rownames_to_column(otu, var = "Query")Execution:
tags <- get_bin_tags(otu)
for_network <- replace_colnames(otu)## Warning in row_to_names(., row_number = 1): Row 1 does not provide unique names.
## Consider running clean_names() after row_to_names().tags <- get_tags(otu)
for_shared <- replace_colnames(otu)## Warning in row_to_names(., row_number = 1): Row 1 does not provide unique names.
## Consider running clean_names() after row_to_names().Prep data for sumarising per region
mixedmdf <- mdf %>%
mutate(habitat = as.character(habitat)) %>%
mutate(habitat = replace_na(habitat, "Mixed")) %>%
mutate(habitat = gsub(" layer", "", habitat)) %>%
filter(!habitat == "100 m above bottom") %>%
filter(!is.na(lat))
try <- otu %>%
data.matrix() %>%
t() %>%
as.data.frame %>%
rownames_to_column(var = "sample") %>%
merge(mixedmdf, ., by = "sample", all.y = TRUE)
with_row <- column_to_rownames(try, var = "sample")
with_row[,6:ncol(with_row)][with_row[,6:ncol(with_row)] > 0 ] <- 1
with_row <- with_row %>% filter(!is.na(lat))
with_row <- with_row[!rowSums(with_row[,6:ncol(with_row)])==0,]
res.pca <- prcomp(with_row[,6:ncol(with_row)],
center = TRUE, scale. = FALSE)
get_eig(res.pca)## eigenvalue variance.percent cumulative.variance.percent
## Dim.1 1.235636e+02 1.860083e+01 18.60083
## Dim.2 4.133834e+01 6.222927e+00 24.82376
## Dim.3 3.769930e+01 5.675119e+00 30.49888
## Dim.4 3.631158e+01 5.466217e+00 35.96510
## Dim.5 2.106870e+01 3.171608e+00 39.13670
## Dim.6 2.033141e+01 3.060618e+00 42.19732
## Dim.7 1.426110e+01 2.146815e+00 44.34414
## Dim.8 1.287627e+01 1.938348e+00 46.28249
## Dim.9 1.269507e+01 1.911070e+00 48.19356
## Dim.10 1.079537e+01 1.625097e+00 49.81865
## Dim.11 1.029378e+01 1.549589e+00 51.36824
## Dim.12 9.961104e+00 1.499509e+00 52.86775
## Dim.13 9.367197e+00 1.410105e+00 54.27786
## Dim.14 9.019676e+00 1.357790e+00 55.63565
## Dim.15 8.318713e+00 1.252270e+00 56.88792
## Dim.16 8.105968e+00 1.220244e+00 58.10816
## Dim.17 8.056506e+00 1.212798e+00 59.32096
## Dim.18 7.999300e+00 1.204186e+00 60.52514
## Dim.19 7.833405e+00 1.179213e+00 61.70436
## Dim.20 7.688611e+00 1.157416e+00 62.86177
## Dim.21 7.593018e+00 1.143026e+00 64.00480
## Dim.22 7.459436e+00 1.122917e+00 65.12772
## Dim.23 7.397463e+00 1.113588e+00 66.24130
## Dim.24 7.249856e+00 1.091368e+00 67.33267
## Dim.25 7.128307e+00 1.073070e+00 68.40574
## Dim.26 7.104494e+00 1.069485e+00 69.47523
## Dim.27 6.996115e+00 1.053170e+00 70.52840
## Dim.28 6.936785e+00 1.044239e+00 71.57264
## Dim.29 6.902948e+00 1.039145e+00 72.61178
## Dim.30 6.660621e+00 1.002666e+00 73.61445
## Dim.31 6.501130e+00 9.786571e-01 74.59310
## Dim.32 6.407848e+00 9.646147e-01 75.55772
## Dim.33 6.306231e+00 9.493177e-01 76.50704
## Dim.34 6.256552e+00 9.418392e-01 77.44888
## Dim.35 6.210241e+00 9.348676e-01 78.38374
## Dim.36 6.112079e+00 9.200906e-01 79.30383
## Dim.37 5.929396e+00 8.925902e-01 80.19642
## Dim.38 5.763768e+00 8.676572e-01 81.06408
## Dim.39 5.631673e+00 8.477721e-01 81.91185
## Dim.40 5.450972e+00 8.205700e-01 82.73242
## Dim.41 5.389881e+00 8.113736e-01 83.54380
## Dim.42 5.323411e+00 8.013674e-01 84.34516
## Dim.43 5.267317e+00 7.929232e-01 85.13809
## Dim.44 5.056815e+00 7.612351e-01 85.89932
## Dim.45 4.881622e+00 7.348621e-01 86.63418
## Dim.46 4.844067e+00 7.292086e-01 87.36339
## Dim.47 4.763941e+00 7.171468e-01 88.08054
## Dim.48 4.637051e+00 6.980453e-01 88.77859
## Dim.49 4.560209e+00 6.864777e-01 89.46506
## Dim.50 4.440668e+00 6.684825e-01 90.13355
## Dim.51 4.281621e+00 6.445401e-01 90.77809
## Dim.52 4.097152e+00 6.167707e-01 91.39486
## Dim.53 3.960984e+00 5.962724e-01 91.99113
## Dim.54 3.905791e+00 5.879640e-01 92.57909
## Dim.55 3.783217e+00 5.695120e-01 93.14860
## Dim.56 3.588145e+00 5.401466e-01 93.68875
## Dim.57 3.485210e+00 5.246511e-01 94.21340
## Dim.58 3.460860e+00 5.209855e-01 94.73439
## Dim.59 3.422107e+00 5.151519e-01 95.24954
## Dim.60 3.350430e+00 5.043619e-01 95.75390
## Dim.61 3.237250e+00 4.873241e-01 96.24123
## Dim.62 3.000120e+00 4.516274e-01 96.69285
## Dim.63 2.251840e+00 3.389840e-01 97.03184
## Dim.64 2.221142e+00 3.343628e-01 97.36620
## Dim.65 2.051549e+00 3.088329e-01 97.67503
## Dim.66 1.950424e+00 2.936099e-01 97.96864
## Dim.67 1.563194e+00 2.353176e-01 98.20396
## Dim.68 1.406802e+00 2.117750e-01 98.41574
## Dim.69 1.358941e+00 2.045701e-01 98.62031
## Dim.70 1.274400e+00 1.918436e-01 98.81215
## Dim.71 1.101545e+00 1.658226e-01 98.97797
## Dim.72 1.001609e+00 1.507787e-01 99.12875
## Dim.73 8.549059e-01 1.286945e-01 99.25745
## Dim.74 8.429984e-01 1.269020e-01 99.38435
## Dim.75 7.747584e-01 1.166294e-01 99.50098
## Dim.76 7.136975e-01 1.074375e-01 99.60841
## Dim.77 6.076474e-01 9.147308e-02 99.69989
## Dim.78 5.186211e-01 7.807138e-02 99.77796
## Dim.79 4.197801e-01 6.319220e-02 99.84115
## Dim.80 3.859904e-01 5.810562e-02 99.89926
## Dim.81 3.474255e-01 5.230020e-02 99.95156
## Dim.82 3.218061e-01 4.844355e-02 100.00000
## Dim.83 2.292289e-25 3.450731e-26 100.00000fviz_eig(res.pca)
p <- fviz_pca_ind(res.pca,
label = "none",
alpha.ind = 0) + scale_color_brewer(palette = "Set2") + geom_point(aes(color = with_row$habitat, shape = with_row$tag, size = 3, alpha = 1)) + theme(text = element_text(size = 20)) + guides(size = "none", color = "legend", alpha = "none", shape = "legend") + labs(shape = "Region", color = "Habitat") + scale_shape_manual(values = c(4,0,2))
p
ggsave(p, filename = "pcatest", device = "png", height = 7, width = 12)# sum the values per row
summed <-
data.matrix(for_shared[,-1]) %>% t(.) %>%
rowsum(., group = sub("\\.\\d+$", "", rownames(.))) %>%
t() %>%
data.frame %>% `rownames<-`(for_shared[,1]) %>%
rownames_to_column(var = "Query")
plotdf <- melt(summed, id.vars = "Query", value.name = "total_present")
# make dummy: 0=not found & 1 = found
plotdf$dummy <- ifelse(plotdf$total_present > 0, yes=1, no=0)
plotdfcomb <- plotdf %>%
group_by(variable) %>%
mutate(presence = if_else(dummy == 1,
ifelse(variable == 'Saba.North', 'SB North',
ifelse(variable == 'Saba.South', 'SB South', 'Statia')),
NULL)) %>%
na.omit() %>%
group_by(Query) %>%
summarise(presence = paste(presence, collapse = ' & '), .groups = 'drop') %>%
mutate(shortpresence = ifelse(presence == 'SB North & SB South & Statia', 'All',
ifelse(presence == 'SB North & SB South', 'SB North & South',
presence)))
pres <-
data.frame(table(plotdfcomb$shortpresence)) %>%
setNames(c('Region', 'Freq')) %>%
arrange(desc(Freq))
pres$Region<- factor(pres$Region, levels = reorder(pres$Region, -pres$Freq))
pres %>%
ggplot() +
geom_bar(aes(x = Region,
y = Freq,
fill = Region),
stat = 'identity') +
theme_minimal() +
theme(axis.text.x = element_text(angle = 45, hjust = 1, size = '10'),
legend.title = element_blank()) +
labs(title = 'Regions where OTUs was detected',
x = 'Region',
y = 'Number of OTUs') +
scale_fill_jco() 
Region plot
lat = c(17.25, 17.65, 17.5, NA, NA, NA, NA)
long = c(-63.4, -63.55, -63.0, NA, NA, NA, NA)
lat_text <- c(NA, NA, NA, mean(c(17.25, 17.65)), mean(c(17.25, 17.65, 17.5)), mean(c(17.65, 17.5))+0.04, mean(c(17.25, 17.5))-0.04)
long_text <- c(NA, NA, NA, mean(c(-63.4, -63.55))-0.06, mean(c(-63.4, -63.55, -63.0)), mean(c(-63.55, -63.0)), mean(c(-63.4,-63.0)))
coordinates <- cbind(lat, long, lat_text, long_text)
test <- cbind(pres, coordinates)
northlength <- length(grep("Saba North", colnames(for_shared)))
southlength <- length(grep("Saba South", colnames(for_shared)))
statialength <- length(grep("Statia", colnames(for_shared)))
test$Freq <- round((test$Freq/c(southlength, northlength, statialength,
mean(c(southlength, northlength)),
mean(c(southlength, northlength, statialength)),
mean(c(northlength, statialength)),
mean(c(southlength, statialength)))) * 25, 0)
allregions <- paste("OTUs present in all regions: ", test[5,2])
test <- test %>% filter(!Region == "All")
p <- ggplot(data=test, aes(x0=long, y0=lat, r=Freq/(8*max(Freq)), fill = Region)) +
geom_segment(aes(x=long[1],
y=lat[1],
xend=long[2],
yend=lat[2]), size = test$Freq[4]/250, color = "gray50") +
geom_segment(aes(x=long[2],
y=lat[2],
xend=long[3],
yend=lat[3]), size = test$Freq[5]/250, color = "gray50") +
geom_segment(aes(x=long[3],
y=lat[3],
xend=long[1],
yend=lat[1]), size = test$Freq[6]/250, color = "gray50") +
geom_circle() +
geom_text(data = test, aes(x=long, y=lat, label = paste(Region, "\n", as.character(Freq), sep = ""))) +
geom_text(data = test, aes(x =long_text, y=lat_text, label = Freq)) +
theme_minimal() +
labs(
#title = "OTUs that are shared between regions",
#subtitle = "Circle correspond to number of OTUs found per region\nthickness of line corresponds to number of OTUs shared",
x = "longitude",
y = "latitude") +
theme(text = element_text(size = 20), panel.grid = element_blank(),
panel.grid.minor = element_blank(),
legend.position = "none") +
scale_fill_brewer(type = "qual", palette = "Pastel2") +
annotate(geom = "text", label = allregions, x = Inf, y = -Inf, hjust = 1.3, vjust = -2)
p## Warning: Removed 3 rows containing non-finite values (stat_circle).## Warning: Removed 3 rows containing missing values (geom_text).
## Warning: Removed 3 rows containing missing values (geom_text).
ggsave("regions", p, device = "png")## Saving 7 x 5 in image## Warning: Removed 3 rows containing non-finite values (stat_circle).
## Warning: Removed 3 rows containing missing values (geom_text).
## Warning: Removed 3 rows containing missing values (geom_text).prepare data for summarising per habitat
prep <- function(df){
df %>%
`rownames<-`(NULL) %>%
column_to_rownames(var = "Query") %>%
data.matrix() %>%
t() %>%
as.data.frame() %>%
rownames_to_column(var = "habitat") %>%
filter(!habitat == "Saba.North.100.m.above.bottom") %>%
mutate(habitat = sub("\\.\\d+$", "", habitat)) %>%
filter(!habitat == "Saba.North.100.m.above.bottom")
}
aggr <- function(df){
aggregate(df[,-1], list(habitat = df$habitat), mean) %>%
mutate(habitat = habitat %>%
sub("Saba.South", "SS", .) %>%
sub("Saba.North", "SN", .) %>%
sub(".above.bottom", "ab", .) %>%
sub(".layer", "", .) %>%
gsub("\\.", " ", .) %>%
sub("0 05", "0.05", ., fixed = TRUE)) %>%
column_to_rownames(var = "habitat") %>%
t() %>%
as.data.frame %>%
rownames_to_column(var = "habitat")
}Execution:
try <- prep(for_network)
try[,-1][try[,-1]>0] <- 1
agr <- try %>% aggr()myPca <- function(df){
res.pca <- prcomp(column_to_rownames(df, var = "habitat"),
center = TRUE, scale. = TRUE)
print(get_eig(res.pca))
fviz_eig(res.pca)
fviz_pca_biplot(res.pca,
label = "var",
col.var = "contrib",
gradient.cols = c("#00AFBB", "#E7B800", "#FC4E07"),
col.ind = "gray80",
repel = TRUE,
geom.ind = "point",
geom.var = "text",
labelsize = 6) + theme_minimal() + theme(text = element_text(size = 20))
}
p_agr <- myPca(agr) +
labs(title = "Presence-absence per bin",
subtitle = "includes all OTUs") + xlim(-12.5, 12.5)## eigenvalue variance.percent cumulative.variance.percent
## Dim.1 3.4579667 31.436061 31.43606
## Dim.2 2.7160396 24.691269 56.12733
## Dim.3 0.9972303 9.065730 65.19306
## Dim.4 0.9955104 9.050094 74.24315
## Dim.5 0.7590921 6.900837 81.14399
## Dim.6 0.5116063 4.650966 85.79496
## Dim.7 0.4595809 4.178008 89.97297
## Dim.8 0.3532729 3.211572 93.18454
## Dim.9 0.2794858 2.540780 95.72532
## Dim.10 0.2540913 2.309921 98.03524
## Dim.11 0.2161238 1.964762 100.00000p_agr
ggsave("finalpcapresence", p_agr, device = "png")## Saving 7 x 5 in image3D Plot
library(geoviz)
#for rgdal, libgdal-dev and libproj-dev were installed to the system first
library(rgdal)## Loading required package: sp## rgdal: version: 1.5-12, (SVN revision 1018)
## Geospatial Data Abstraction Library extensions to R successfully loaded
## Loaded GDAL runtime: GDAL 2.2.2, released 2017/09/15
## Path to GDAL shared files: /usr/share/gdal/2.2
## GDAL binary built with GEOS: TRUE
## Loaded PROJ runtime: Rel. 4.9.2, 08 September 2015, [PJ_VERSION: 492]
## Path to PROJ shared files: (autodetected)
## Linking to sp version:1.4-2library(rasterVis)## Loading required package: raster##
## Attaching package: 'raster'## The following object is masked _by_ '.GlobalEnv':
##
## getData## The following object is masked from 'package:janitor':
##
## crosstab## The following object is masked from 'package:data.table':
##
## shift## The following object is masked from 'package:tidyr':
##
## extract## The following object is masked from 'package:dplyr':
##
## select## Loading required package: lattice## Loading required package: latticeExtra##
## Attaching package: 'latticeExtra'## The following object is masked from 'package:ggplot2':
##
## layerlibrary(rgl)
library(scatterplot3d)
library(raster)
library(plotly)##
## Attaching package: 'plotly'## The following object is masked from 'package:raster':
##
## select## The following object is masked from 'package:ggplot2':
##
## last_plot## The following objects are masked from 'package:plyr':
##
## arrange, mutate, rename, summarise## The following object is masked from 'package:stats':
##
## filter## The following object is masked from 'package:graphics':
##
## layout# get points for samples
Point <- mdf %>% select(long, lat, altitude) %>% as.matrix()
colnames(Point) <- c("x", "y", "z")
rownames(Point) <- c(1:nrow(Point))
# get width of the map
Point.extent <- extent(Point[,1:2])
# get elevation file into a raster
elev.new1 <- raster::raster("~/Downloads/gebco_2020_n17.8_s17.1_w-63.7_e-62.9.tif")
# get dimensions of the elevation file
d <- dim(elev.new1)
# make a new raster that has same row, col, extent and crs
point.raster <- raster(nrow = nrow(elev.new1), ncol=ncol(elev.new1), extent(elev.new1))
crs(point.raster) <- crs(elev.new1)
cells <- cellFromXY(point.raster, Point[,1:2])
point.raster[cells] <- Point[,3]
# flip around elev file so the mountains/valleys are correct
elev.new2 <- t(as.matrix(elev.new1))
#get row and col number using the cell number, and set it to df
#point.raster2 <- as.matrix(point.raster)
temp <- as.data.frame(cbind(cells, altitude = Point[,3]))
point.df <- data.frame(rowColFromCell(point.raster,
as.numeric(temp$cells)),
altitude = temp$altitude)
samples_co_3d <- cbind(mdf, point.df)[,-4]# get otu, change query col to rownames, make all values above 0 one
otu[otu > 0] <- 1
# Number of OTUs that are present in one sample
length(rowSums(otu)==1)## [1] 17948# filter out where otu is found in only 1 sample
otu <- otu[rowSums(otu) > 1,]
otu <- rownames_to_column(otu, var = "Query")
# merge the otu file and the plotdf
merged <- merge(otu, plotdf, by = "Query")
# function to get all possible combinations for samples were a single OTU is present
getCombsCoNames <- function(r, df){
mySamples <- colnames(df)[df[r,] == 1]
myCombsM <- combn(mySamples, 2)
transmyCombsM <- t(myCombsM)
df <- as.data.frame(transmyCombsM)}
togetcombs <- merged %>%
`rownames<-`(NULL) %>% select(!Query)
# have a dataframe with all names combinations (takes +- 16 secs)
allCombsCoNames <- lapply(1:nrow(togetcombs), getCombsCoNames, df=togetcombs)
length(allCombsCoNames)## [1] 25248#add group ID because it needs to be ungrouped later
Names_add_id <- lapply(1:length(allCombsCoNames), function(x)
cbind(allCombsCoNames[[x]],
id=rep(as.character(x),nrow(allCombsCoNames[[x]]))))
# bind the dfs by row
Names_merged <- rbindlist(Names_add_id)
# remove duplicate rows and split into seperate dfs again
# because it takes a lot of time to add lines between points that are already drawn and it doenst add value
# idea is to record how many times a combination occurs so to adjust the thickness of the length
Combs_unique <-
Names_merged %>%
dplyr::distinct(V1, V2, .keep_all=TRUE) %>%
group_by(id) %>%
group_split(.keep=F)
combs_unique_matrix <- lapply(Combs_unique, as.matrix)
combs_unique_tmatrix <- lapply(combs_unique_matrix, t)
# now we have a list of matrices
# unname them and transform into list
# now there are many list. For every OTU that occurs at two location there is listed what locatios it is connected to.
lines <- lapply(combs_unique_tmatrix, function(x){
m <- unname(x)
l <- unlist(apply(m, 2, list))
samples_co_3d[match(l, samples_co_3d$sample),]})
#max 7832 combinations possible in total based on all samples
length(combn(samples,2))## [1] 7832# if needed take random sample of lines
lines_sample <- sample(lines, 50)Generate plot
library(plotly)
plot3D <- function(lines){
p <- plot_ly(z = elev.new2, type = "surface",
alpha = 1, size = 10, opacity=0.9,
colors = colorRamp(c("black", "midnightblue","darkblue", "steelblue",
"steelblue1", "wheat", "seagreen1","seagreen"),
bias = 0.9),
scene = 'scene2',
surfaceaxis = -1 ) %>%
#hide the colorscale bar
hide_colorbar() %>%
#add Saba North scatter points
add_trace(data = samples_co_3d[samples_co_3d$tag=='Saba North',],
x = ~row, y=~col, z=~altitude,
mode = "markers", type = "scatter3d",
name = "Saba Bank North",
markers = list(symbol = 0,
color = rgb(0.93, 0.68, 0.05, alpha = 0.5), size = 2.5,
line = list(color=rgb(0.0, 0.0, 0), width = 0.5))) %>%
# " Saba South "
add_trace(data = samples_co_3d[samples_co_3d$tag=='Saba South',],
x = ~row, y=~col, z=~altitude,
mode = "markers", type = "scatter3d",
name = "Saba Bank South",
markers = list(symbol = 0,
color = rgb(0.94, 0.50, 0.50, alpha = 0.5), size = 2.5,
line = list(color=rgb(0.0, 0.0, 0), width = 0.5))) %>%
# " Statia "
add_trace(data = samples_co_3d[samples_co_3d$tag=='Statia',],
x = ~row, y=~col, z=~altitude,
mode = "markers",
type = "scatter3d",
name = "Statia",
markers = list(symbol = 0, color = rgb(1, 0.27, 0, alpha = 0.5),
size = 2.5,
line = list(color=rgb(0.0, 0.0, 0), width = 0.5))) %>%
layout(title = "3D bathymetric map of sampling area with sampling points",
legend=list(
#itemsizing = "constant",
annotations = list(yref = "paper",
xref="paper",
y = 1.05, x=1.1,
showarrow =F,
text = "Region")),
showlegend = T)
if (lines){
for(df in lines_sample){
p <- add_trace(p,
x = df$row,y = df$col,
z = df$altitude,
type = "scatter3d",
mode = 'lines',
showlegend = F,
line = list(width = 1))
}}
return(p)
}
# make plot with lines
p <- plot3D(lines=T)## Warning: `arrange_()` is deprecated as of dplyr 0.7.0.
## Please use `arrange()` instead.
## See vignette('programming') for more help
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_warnings()` to see where this warning was generated.## Warning: 'surface' objects don't have these attributes: 'surfaceaxis'
## Valid attributes include:
## 'type', 'visible', 'legendgroup', 'name', 'uid', 'ids', 'customdata', 'meta', 'hoverlabel', 'stream', 'uirevision', 'z', 'x', 'y', 'text', 'hovertext', 'hovertemplate', 'connectgaps', 'surfacecolor', 'cauto', 'cmin', 'cmax', 'cmid', 'colorscale', 'autocolorscale', 'reversescale', 'showscale', 'colorbar', 'coloraxis', 'contours', 'hidesurface', 'lightposition', 'lighting', 'opacity', '_deprecated', 'hoverinfo', 'showlegend', 'xcalendar', 'ycalendar', 'zcalendar', 'scene', 'idssrc', 'customdatasrc', 'metasrc', 'zsrc', 'xsrc', 'ysrc', 'textsrc', 'hovertextsrc', 'hovertemplatesrc', 'surfacecolorsrc', 'hoverinfosrc', 'key', 'set', 'frame', 'transforms', '_isNestedKey', '_isSimpleKey', '_isGraticule', '_bbox'p## Warning: 'surface' objects don't have these attributes: 'surfaceaxis'
## Valid attributes include:
## 'type', 'visible', 'legendgroup', 'name', 'uid', 'ids', 'customdata', 'meta', 'hoverlabel', 'stream', 'uirevision', 'z', 'x', 'y', 'text', 'hovertext', 'hovertemplate', 'connectgaps', 'surfacecolor', 'cauto', 'cmin', 'cmax', 'cmid', 'colorscale', 'autocolorscale', 'reversescale', 'showscale', 'colorbar', 'coloraxis', 'contours', 'hidesurface', 'lightposition', 'lighting', 'opacity', '_deprecated', 'hoverinfo', 'showlegend', 'xcalendar', 'ycalendar', 'zcalendar', 'scene', 'idssrc', 'customdatasrc', 'metasrc', 'zsrc', 'xsrc', 'ysrc', 'textsrc', 'hovertextsrc', 'hovertemplatesrc', 'surfacecolorsrc', 'hoverinfosrc', 'key', 'set', 'frame', 'transforms', '_isNestedKey', '_isSimpleKey', '_isGraticule', '_bbox'## Warning: 'scatter3d' objects don't have these attributes: 'markers'
## Valid attributes include:
## 'type', 'visible', 'showlegend', 'legendgroup', 'opacity', 'name', 'uid', 'ids', 'customdata', 'meta', 'hoverlabel', 'stream', 'transforms', 'uirevision', 'x', 'y', 'z', 'text', 'texttemplate', 'hovertext', 'hovertemplate', 'mode', 'surfaceaxis', 'surfacecolor', 'projection', 'connectgaps', 'line', 'marker', 'textposition', 'textfont', 'hoverinfo', 'error_x', 'error_y', 'error_z', 'xcalendar', 'ycalendar', 'zcalendar', 'scene', 'idssrc', 'customdatasrc', 'metasrc', 'xsrc', 'ysrc', 'zsrc', 'textsrc', 'texttemplatesrc', 'hovertextsrc', 'hovertemplatesrc', 'textpositionsrc', 'hoverinfosrc', 'key', 'set', 'frame', 'transforms', '_isNestedKey', '_isSimpleKey', '_isGraticule', '_bbox'
## Warning: 'scatter3d' objects don't have these attributes: 'markers'
## Valid attributes include:
## 'type', 'visible', 'showlegend', 'legendgroup', 'opacity', 'name', 'uid', 'ids', 'customdata', 'meta', 'hoverlabel', 'stream', 'transforms', 'uirevision', 'x', 'y', 'z', 'text', 'texttemplate', 'hovertext', 'hovertemplate', 'mode', 'surfaceaxis', 'surfacecolor', 'projection', 'connectgaps', 'line', 'marker', 'textposition', 'textfont', 'hoverinfo', 'error_x', 'error_y', 'error_z', 'xcalendar', 'ycalendar', 'zcalendar', 'scene', 'idssrc', 'customdatasrc', 'metasrc', 'xsrc', 'ysrc', 'zsrc', 'textsrc', 'texttemplatesrc', 'hovertextsrc', 'hovertemplatesrc', 'textpositionsrc', 'hoverinfosrc', 'key', 'set', 'frame', 'transforms', '_isNestedKey', '_isSimpleKey', '_isGraticule', '_bbox'
## Warning: 'scatter3d' objects don't have these attributes: 'markers'
## Valid attributes include:
## 'type', 'visible', 'showlegend', 'legendgroup', 'opacity', 'name', 'uid', 'ids', 'customdata', 'meta', 'hoverlabel', 'stream', 'transforms', 'uirevision', 'x', 'y', 'z', 'text', 'texttemplate', 'hovertext', 'hovertemplate', 'mode', 'surfaceaxis', 'surfacecolor', 'projection', 'connectgaps', 'line', 'marker', 'textposition', 'textfont', 'hoverinfo', 'error_x', 'error_y', 'error_z', 'xcalendar', 'ycalendar', 'zcalendar', 'scene', 'idssrc', 'customdatasrc', 'metasrc', 'xsrc', 'ysrc', 'zsrc', 'textsrc', 'texttemplatesrc', 'hovertextsrc', 'hovertemplatesrc', 'textpositionsrc', 'hoverinfosrc', 'key', 'set', 'frame', 'transforms', '_isNestedKey', '_isSimpleKey', '_isGraticule', '_bbox'#To save plot to file:
#library(htmlwidgets)
#saveWidget(p, file = "3Dplot_lines.html", selfcontained = T)